Single-genome sequencing reveals within-host evolution of human malaria parasites.

Population genomics of bulk malaria infections is unable to examine intrahost evolution; therefore, most work has focused on the role of recombination in generating genetic variation. We used single-cell sequencing protocol for low-parasitaemia infections to generate 406 near-complete single Plasmodium vivax genomes from 11 patients sampled during sequential febrile episodes. Parasite genomes contain hundreds of de novo mutations, showing strong signatures of selection, which are enriched in the ApiAP2 family of transcription factors, known targets of adaptation. Comparing 315 P. falciparum single-cell genomes from 15 patients with our P. vivax data, we find broad complementary patterns of de novo mutation at the gene and pathway level, revealing the importance of within-host evolution during malaria infections.

The power and promise of genetic mapping from Plasmodium falciparum crosses utilizing human liver-chimeric mice.

Genetic crosses are most powerful for linkage analysis when progeny numbers are high, parental alleles segregate evenly and numbers of inbred progeny are minimized. We previously developed a novel genetic crossing platform for the human malaria parasite Plasmodium falciparum, an obligately sexual, hermaphroditic protozoan, using mice carrying human hepatocytes (the human liver-chimeric FRG NOD huHep mouse) as the vertebrate host. We report on two genetic crosses-(1) an allopatric cross between a laboratory-adapted parasite (NF54) of African origin and a recently patient-derived Asian parasite, and (2) a sympatric cross between two recently patient-derived Asian parasites. We generated 144 unique recombinant clones from the two crosses, doubling the number of unique recombinant progeny generated in the previous 30 years. The allopatric African/Asian cross has minimal levels of inbreeding and extreme segregation distortion, while in the sympatric Asian cross, inbred progeny predominate and parental alleles segregate evenly. Using simulations, we demonstrate that these progeny provide the power to map small-effect mutations and epistatic interactions. The segregation distortion in the allopatric cross slightly erodes power to detect linkage in several genome regions. We greatly increase the power and the precision to map biomedically important traits with these new large progeny panels.

Co-transmission of Related Malaria Parasite Lineages Shapes Within-Host Parasite Diversity.

In high-transmission regions, we expect parasite lineages within complex malaria infections to be unrelated due to parasite inoculations from different mosquitoes. This project was designed to test this prediction. We generated 485 single-cell genome sequences from fifteen P. falciparum malaria patients from Chikhwawa, Malawi-an area of intense transmission. Patients harbored up to seventeen unique parasite lineages. Surprisingly, parasite lineages within infections tend to be closely related, suggesting that superinfection by repeated mosquito bites is rarer than co-transmission of parasites from a single mosquito. Both closely and distantly related parasites comprise an infection, suggesting sequential transmission of complex infections between multiple hosts. We identified tetrads and reconstructed parental haplotypes, which revealed the inbred ancestry of infections and non-Mendelian inheritance. Our analysis suggests strong barriers to secondary infection and outbreeding amongst malaria parasites from a high transmission setting, providing unexpected insights into the biology and transmission of malaria.

Outcomes of a Care Coordination Guardianship Intervention for Adults with Severe Mental Illness: An Interrupted Time Series Analysis.

This study investigates the impacts of an intensive care coordination intervention for adults with severe mental illness who have guardians. Program impacts on medical and legal outcomes were assessed using interrupted time series analysis. Program participation was associated with statistically significant reductions in the level and trend of psychiatric hospitalizations, number of days in psychiatric hospitals, emergency room visits, and arrests. Days incarcerated did not change significantly. The decrease in medical and legal outcomes may be associated with decreased spending on these services. The program and other intensive care coordination interventions for this population warrant further replication and research.

[18F]-Fluorodeoxyglucose Uptake in Lymphoid Tissue Serves as a Predictor of Disease Outcome in the Nonhuman Primate Model of Monkeypox Virus Infection.

Real-time bioimaging of infectious disease processes may aid countermeasure development and lead to an improved understanding of pathogenesis. However, few studies have identified biomarkers for monitoring infections using in vivo imaging. Previously, we demonstrated that positron emission tomography/computed tomography (PET/CT) imaging with [18F]-fluorodeoxyglucose (FDG) can monitor monkeypox disease progression in vivo in nonhuman primates (NHPs). In this study, we investigated [18F]-FDG-PET/CT imaging of immune processes in lymphoid tissues to identify patterns of inflammation in the monkepox NHP model and to determine the value of [18F]-FDG-PET/CT as a biomarker for disease and treatment outcomes. Quantitative analysis of [18F]-FDG-PET/CT images revealed differences between moribund and surviving animals at two sites vital to the immune response to viral infections, bone marrow and lymph nodes (LNs). Moribund NHPs demonstrated increased [18F]-FDG uptake in bone marrow 4 days postinfection compared to surviving NHPs. In surviving, treated NHPs, increase in LN volume correlated with [18F]-FDG uptake and peaked 10 days postinfection, while minimal lymphadenopathy and higher glycolytic activity were observed in moribund NHPs early in infection. Imaging data were supported by standard virology, pathology, and immunology findings. Even with the limited number of subjects, imaging was able to differentiate the difference between disease outcomes, warranting additional studies to demonstrate whether [18F]-FDG-PET/CT can identify other, subtler effects. Visualizing altered metabolic activity at sites involved in the immune response by [18F]-FDG-PET/CT imaging is a powerful tool for identifying key disease-specific time points and locations that are most relevant for pathogenesis and treatment.IMPORTANCE Positron emission tomography and computed tomography (PET/CT) imaging is a universal tool in oncology and neuroscience. The application of this technology to infectious diseases is far less developed. We used PET/CT imaging with [18F]-labeled fluorodeoxyglucose ([18F]-FDG) in monkeys after monkeypox virus exposure to monitor the immune response in lymphoid tissues. In lymph nodes of surviving monkeys, changes in [18F]-FDG uptake positively correlated with enlargement of the lymph nodes and peaked on day 10 postinfection. In contrast, the bone marrow and lymph nodes of nonsurvivors showed increased [18F]-FDG uptake by day 4 postinfection with minimal lymph node enlargement, indicating that elevated cell metabolic activity early after infection is predictive of disease outcome. [18F]-FDG-PET/CT imaging can provide real-time snapshots of metabolic activity changes in response to viral infections and identify key time points and locations most relevant for monitoring the development of pathogenesis and for potential treatment to be effective.

Loss in lung volume and changes in the immune response demonstrate disease progression in African green monkeys infected by small-particle aerosol and intratracheal exposure to Nipah virus.

Nipah virus (NiV) is a paramyxovirus (genus Henipavirus) that emerged in the late 1990s in Malaysia and has since been identified as the cause of sporadic outbreaks of severe febrile disease in Bangladesh and India. NiV infection is frequently associated with severe respiratory or neurological disease in infected humans with transmission to humans through inhalation, contact or consumption of NiV contaminated foods. In the work presented here, the development of disease was investigated in the African Green Monkey (AGM) model following intratracheal (IT) and, for the first time, small-particle aerosol administration of NiV. This study utilized computed tomography (CT) and magnetic resonance imaging (MRI) to temporally assess disease progression. The host immune response and changes in immune cell populations over the course of disease were also evaluated. This study found that IT and small-particle administration of NiV caused similar disease progression, but that IT inoculation induced significant congestion in the lungs while disease following small-particle aerosol inoculation was largely confined to the lower respiratory tract. Quantitative assessment of changes in lung volume found up to a 45% loss in IT inoculated animals. None of the subjects in this study developed overt neurological disease, a finding that was supported by MRI analysis. The development of neutralizing antibodies was not apparent over the 8-10 day course of disease, but changes in cytokine response in all animals and activated CD8+ T cell numbers suggest the onset of cell-mediated immunity. These studies demonstrate that IT and small-particle aerosol infection with NiV in the AGM model leads to a severe respiratory disease devoid of neurological indications. This work also suggests that extending the disease course or minimizing the impact of the respiratory component is critical to developing a model that has a neurological component and more accurately reflects the human condition.

Exposure of rhesus monkeys to cowpox virus Brighton Red by large-particle aerosol droplets results in an upper respiratory tract disease.

We previously demonstrated that small-particle (0.5-3.0 µm) aerosol infection of rhesus monkeys (Macaca mulatta) with cowpox virus (CPXV)-Brighton Red (BR) results in fulminant respiratory tract disease characterized by severe lung parenchymal pathology but only limited systemic virus dissemination and limited classic epidermal pox-like lesion development (Johnson et al., 2015). Based on these results, and to further develop CPXV as an improved model of human smallpox, we evaluated a novel large-particle aerosol (7.0-9.0 µm) exposure of rhesus monkeys to CPXV-BR and monitored for respiratory tract disease by serial computed tomography (CT). As expected, the upper respiratory tract and large airways were the major sites of virus-induced pathology following large-particle aerosol exposure. Large-particle aerosol CPXV exposure of rhesus macaques resulted in severe upper airway and large airway pathology with limited systemic dissemination.

Small particle aerosol inoculation of cowpox Brighton Red in rhesus monkeys results in a severe respiratory disease.

Cowpox virus (CPXV) inoculation of nonhuman primates (NHPs) has been suggested as an alternate model for smallpox (Kramski et al., 2010, PLoS One, 5, e10412). Previously, we have demonstrated that intrabronchial inoculation of CPXV-Brighton Red (CPXV-BR) into cynomolgus monkeys resulted in a disease that shared many similarities to smallpox; however, severe respiratory tract disease was observed (Smith et al., 2011, J. Gen. Virol.). Here we describe the course of disease after small particle aerosol exposure of rhesus monkeys using computed tomography (CT) to monitor respiratory disease progression. Subjects developed a severe respiratory disease that was uniformly lethal at 5.7 log10 PFU of CPXV-BR. CT indicated changes in lung architecture that correlated with changes in peripheral blood monocytes and peripheral oxygen saturation. While the small particle aerosol inoculation route does not accurately mimic human smallpox, the data suggest that CT can be used as a tool to monitor real-time disease progression for evaluation of animal models for human diseases.

Monkeypox virus infection of rhesus macaques induces massive expansion of natural killer cells but suppresses natural killer cell functions.

Natural killer (NK) cells play critical roles in innate immunity and in bridging innate and adaptive immune responses against viral infection. However, the response of NK cells to monkeypox virus (MPXV) infection is not well characterized. In this intravenous challenge study of MPXV infection in rhesus macaques (Macaca mulatta), we analyzed blood and lymph node NK cell changes in absolute cell numbers, cell proliferation, chemokine receptor expression, and cellular functions. Our results showed that the absolute number of total NK cells in the blood increased in response to MPXV infection at a magnitude of 23-fold, manifested by increases in CD56+, CD16+, CD16-CD56- double negative, and CD16+CD56+ double positive NK cell subsets. Similarly, the frequency and NK cell numbers in the lymph nodes also largely increased with the total NK cell number increasing 46.1-fold. NK cells both in the blood and lymph nodes massively proliferated in response to MPXV infection as measured by Ki67 expression. Chemokine receptor analysis revealed reduced expression of CXCR3, CCR7, and CCR6 on NK cells at early time points (days 2 and 4 after virus inoculation), followed by an increased expression of CXCR3 and CCR5 at later time points (days 7-8) of infection. In addition, MPXV infection impaired NK cell degranulation and ablated secretion of interferon-γ and tumor necrosis factor-α. Our data suggest a dynamic model by which NK cells respond to MPXV infection of rhesus macaques. Upon virus infection, NK cells proliferated robustly, resulting in massive increases in NK cell numbers. However, the migrating capacity of NK cells to tissues at early time points might be reduced, and the functions of cytotoxicity and cytokine secretion were largely compromised. Collectively, the data may explain, at least partially, the pathogenesis of MPXV infection in rhesus macaques.

Poxvirus antigen staining of immune cells as a biomarker to predict disease outcome in monkeypox and cowpox virus infection in non-human primates.

Infection of non-human primates (NHPs) such as rhesus and cynomolgus macaques with monkeypox virus (MPXV) or cowpox virus (CPXV) serve as models to study poxvirus pathogenesis and to evaluate vaccines and anti-orthopox therapeutics. Intravenous inoculation of macaques with high dose of MPXV (>1-2×10(7) PFU) or CPXV (>10(2) PFU) results in 80% to 100% mortality and 66 to 100% mortality respectively. Here we report that NHPs with positive detection of poxvirus antigens in immune cells by flow cytometric staining, especially in monocytes and granulocytes succumbed to virus infection and that early positive pox staining is a strong predictor for lethality. Samples from four independent studies were analyzed. Eighteen NHPs from three different experiments were inoculated with two different MPXV strains at lethal doses. Ten NHPs displayed positive pox-staining and all 10 NHPs reached moribund endpoint. In contrast, none of the three NHPs that survived anticipated lethal virus dose showed apparent virus staining in the monocytes and granulocytes. In addition, three NHPs that were challenged with a lethal dose of MPXV and received cidofovir treatment were pox-antigen negative and all three NHPs survived. Furthermore, data from a CPXV study also demonstrated that 6/9 NHPs were pox-antigen staining positive and all 6 NHPs reached euthanasia endpoint, while the three survivors were pox-antigen staining negative. Thus, we conclude that monitoring pox-antigen staining in immune cells can be used as a biomarker to predict the prognosis of virus infection. Future studies should focus on the mechanisms and implications of the pox-infection of immune cells and the correlation between pox-antigen detection in immune cells and disease progression in human poxviral infection.

Simian hemorrhagic fever virus infection of rhesus macaques as a model of viral hemorrhagic fever: clinical characterization and risk factors for severe disease.

Simian Hemorrhagic Fever Virus (SHFV) has caused sporadic outbreaks of hemorrhagic fevers in macaques at primate research facilities. SHFV is a BSL-2 pathogen that has not been linked to human disease; as such, investigation of SHFV pathogenesis in non-human primates (NHPs) could serve as a model for hemorrhagic fever viruses such as Ebola, Marburg, and Lassa viruses. Here we describe the pathogenesis of SHFV in rhesus macaques inoculated with doses ranging from 50 PFU to 500,000 PFU. Disease severity was independent of dose with an overall mortality rate of 64% with signs of hemorrhagic fever and multiple organ system involvement. Analyses comparing survivors and non-survivors were performed to identify factors associated with survival revealing differences in the kinetics of viremia, immunosuppression, and regulation of hemostasis. Notable similarities between the pathogenesis of SHFV in NHPs and hemorrhagic fever viruses in humans suggest that SHFV may serve as a suitable model of BSL-4 pathogens.

Comparative analysis of monkeypox virus infection of cynomolgus macaques by the intravenous or intrabronchial inoculation route.

Monkeypox virus (MPXV) infection has recently expanded in geographic distribution and can be fatal in up to 10% of cases. The intravenous (i.v.) inoculation of nonhuman primates (NHPs) results in an accelerated fulminant disease course compared to that of naturally occurring MPXV infection in humans. Alternative routes of inoculation are being investigated to define an NHP model of infection that more closely resembles natural disease progression. Our goal was to determine if the intrabronchial (i.b.) exposure of NHPs to MPXV results in a systemic disease that better resembles the progression of human MPXV infection. Here, we compared the disease course following an i.v. or i.b. inoculation of NHPs with 10-fold serial doses of MPXV Zaire. Classical pox-like disease was observed in NHPs administered a high virus dose by either route. Several key events were delayed in the highest doses tested of the i.b. model compared to the timing of the i.v. model, including the onset of fever, lesion appearance, peak viremia, viral shedding in nasal and oral swabs, peak cytokine levels, and time to reach endpoint criteria. Virus distribution across 19 tissues was largely unaffected by the inoculation route at the highest doses tested. The NHPs inoculated by the i.b. route developed a viral pneumonia that likely exacerbated disease progression. Based on the observations of the delayed onset of clinical and virological parameters and endpoint criteria that may more closely resemble those of human MPXV infection, the i.b. MPXV model should be considered for the further investigation of viral pathogenesis and countermeasures.

Arterial endothelial dysfunction in baboons fed a high-cholesterol, high-fat diet.

BACKGROUND: Endothelial dysfunction signals the initiation and progression of atherosclerosis. Elevated LDL-cholesterol concentrations have been suggested to induce endothelial dysfunction, but direct in vivo evidence for the relation is still lacking. OBJECTIVE: We examined the hypothesis that a high-cholesterol, high-fat (HCHF) diet can directly cause endothelial dysfunction in vivo. DESIGN: We measured inflammatory and endothelial dysfunctional markers in circulating blood and directly in endothelial cells, which were collected by femoral artery biopsies, in 10 baboons before and after a 7-wk HCHF dietary challenge. RESULTS: We found that the HCHF diet induced a high inflammatory status, as indicated by increased concentrations of interleukin 6, tumor necrosis factor alpha (TNF-alpha), and monocyte chemoattractant protein 1. Although the concentrations of endothelial dysfunctional markers, such as soluble vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1, were not increased by the HCHF diet, membrane-bound VCAM-1 and membrane-bound E-selectin on endothelial cells were highly increased after 7 wk of the HCHF diet (P < 0.01). In contrast, the concentrations of endothelial nitric oxide synthase in endothelial cells were significantly reduced by the 7-wk HCHF diet (P < 0.01). Furthermore, the dietary challenge attenuated endothelial cell responses to TNF-alpha, lipopolysaccharide, native LDL cholesterol, and oxidized LDL-cholesterol stimulation. CONCLUSIONS: Our results show that an HCHF diet can directly induce inflammation and endothelial dysfunction. Prior in vivo exposure to an HCHF diet attenuates the in vitro responses of endothelial cells to atherogenic risk factors. This preconditioning phenomenon may have significant clinical relevance.